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WO1998000434A1 - Oligonucleotides modifies - Google Patents

Oligonucleotides modifies Download PDF

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Publication number
WO1998000434A1
WO1998000434A1 PCT/EP1997/003192 EP9703192W WO9800434A1 WO 1998000434 A1 WO1998000434 A1 WO 1998000434A1 EP 9703192 W EP9703192 W EP 9703192W WO 9800434 A1 WO9800434 A1 WO 9800434A1
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WO
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Prior art keywords
alkyl
methyl
phenyl
mmol
oligonucleotide
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PCT/EP1997/003192
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English (en)
Inventor
Alain De Mesmaeker
Sebastian Wendeborn
Jacques Lebreton
Original Assignee
Novartis Ag
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Publication date
Application filed by Novartis Ag filed Critical Novartis Ag
Priority to AU33407/97A priority Critical patent/AU3340797A/en
Publication of WO1998000434A1 publication Critical patent/WO1998000434A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to modified oligonucieotides comprising at least one nucleotide dimer with a modified backbone, to the modified nucleotide dimers in a certain configuration, processes for the preparation of these oligonucieotides or the nucleotide dimers, the use of these oligonucieotides or the nucleotide dimers and pharmaceutical preparations containing the modified oligonucieotides.
  • Nucleosides and oligonucieotides have acquired wide interest as antiviral active ingredients or because of their capability to interact with nucleic acids ("antisense” oligonucieotides) and the biological activity associated therewith, see, for example, Uhlmann & Peyman, Chemical Reviews (1990), 90, 543-584.
  • antisense oligonucieotides
  • the sugar radicals of nucleosides (or the nucleotide units in oligonucieotides) or the internucleotide phosphate bond in oligonucieotides have been modified in very different ways.
  • the current invention provides oligonucieotides in a certain configuration that are capable of a surprisingly strong hybridization to target RNA or DNA.
  • oligonucleotide of formula 1 comprises at least one structural unit of formula 2 wherein
  • R 1 is H, C 1 -C 4 alkyl or d-dalkoxy; preferred is H or d-C 4 alkyl; more preferred is H or methyl; most preferred is H;
  • R 2 is H, C C 4 alkyl, phenyl, C ⁇ -C 4 alkyl-phenyl, C 3 -C 9 heteroaryl, C ⁇ -C 4 alkyl-C 3 -C 9 heteroaryl or an intercalator; wherein the aryl or heteroaryl is unsubstituted or substituted by OH,
  • R 4 d-dalkoxy, -O-(CH 2 -CH 2 -O) m R 4 , NR 4 2 or NHR 4 ; preferred is H, d-dalkyl, phenyl, CrC 4 alkyl-phenyl or C 3 -C 9 heteroaryl; more preferred is H, methyl, ethyl or phenyl; most preferred is H, methyl or phenyl; R 3 is C C 4 alkyl, unsubstituted or substituted by OH, NR 2 or NHR 4 ; preferred is d-dalkyl; more preferred is methyl or ethyl; most preferred is methyl; R 4 is H or d-C 4 alkyl; preferred is methyl or ethyl; more preferred is methyl; X and Y are independent of one another, H , OH , OR 4 , O-d-C 4 alkylNHR 4 , O-C
  • R 5 is H or C ⁇ -C ⁇ 0 alkyl; preferred is H, CH 3 or d-C 4 alkyl; more preferred is H, methyl or ethyl; R 6 is H, CH 3 or an OH-protecting group; m is an integer from 1 to 4; preferred is 1 ; and A and B are, independent of one another, a purine or pyrimidine radical or an analogue thereof; with the proviso that if A and B are thymidine, R 1 , R 2 and X are hydrogen and Y is methoxy, R 3 is not methyl.
  • the oligonucleotide may be further modified, e.g., by replacement of phosphodiester bonds with -thioate bonds.
  • alkyl, alkoxy, hydroxyalkyl and aminoalkyl are methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl, and also the corresponding alkoxy, hydroxyalkyl and aminoalkyl radicals.
  • the alkyl, alkoxy, hydroxyalkyl and aminoalkyl radicals preferably contain 1 to 4 C atoms like methyl, ethyl, n- and i-propyl, n-, i- and t-butyl, methoxy, ethoxy, aminomethyl, aminoethyl, hydroxymethyl and hydroxyethyl.
  • aminoalkyl examples are also aminomethyl, aminoethyl, 1 -aminoprop-2-yl or -3-yl, 1 -aminobut-2-yl or -3-yl or -4-yl, N-methyl- or N,N-dimethyl- or N-ethyl- or N,N-diethyl- or N-2-hydroxyethyl- or N,N-di-2-hydroxyethylaminomethyl or -aminoethyl or -aminopropyl or -aminobutyl.
  • hydroxyalkyl examples are hydroxymethyl, 1 -hydroxyeth-2-yl, 1 -hydroxy- prop-2- or -3-yl, 1 -hydroxybut-2-yl, -3-yl or -4-yl.
  • C 6 -C ⁇ oaryl examples are naphthyl and phenyl, wherein phenyl is preferred.
  • the heteroaryl preferably contains 1 to 3 heteroatoms selected from the group consisting of O, S and N, like thienyl, furyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl and pyridazinyl.
  • a preferred intercalator in connection with the present invention is anthraquinone substituted by a linker, the linker being preferably a chain of 2 to 7 atoms selected from the group consisting of C, N and O, like C 2 -C 7 alkyi. If A and/or B is a purine radical or an analogue thereof, it can be a radical of the formula 3, 4, 5 or 6.
  • R 16 is as defined below.
  • the primary amino preferably contains 1 to 12 and particularly preferably 1 to 6 C atoms, and the secondary amino preferably 2 to 12 and particularly preferably 2 to 6 C atoms.
  • alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl which preferably contain 1 to 6 C atoms, are methyl, ethyl and the isomers of propyl, butyl, pentyl and hexyl; and also corresponding alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals.
  • the alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals preferably contain 1 to 4 C atoms.
  • Preferred alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals are methyl, ethyl, n- and i-propyl, n-, i- and t-butyl, methoxy, ethoxy, methylthio and ethylthio, aminomethyl, aminoethyl, hydroxymethyl and hydroxyethyl.
  • the primary amino and secondary amino can, for example, be radicals of the formula R 13 R 1 N, in which R 13 and R 14 are independently H, d-C 2 oalkyl, -aminoalkyl or -hydroxyalkyl, preferably d-C 12 alkyl, -aminoalkyl or -hydroxyalkyl and particularly preferably d-C 6 alkyl, - aminoalkyl or -hydroxyalkyl; carboxyalkyl or carbalkoxyalkyl, where the carbalkoxy group contains 2 to 8 C atoms and the alkyl group contains 1 to 6, preferably 1 to 4, C atoms; C 2 - doalkenyl, preferably C 2 -C 12 alkenyl and particularly preferably C 2 -C 6 alkenyl; phenyl, mono- or di(d-C 4 alkyl- or -alkoxy)phenyl, benzyl, mono- or di(d-C 4 alkyl- or -alkoxy)
  • the amino group in the aminoalkyl is unsubstituted or substituted by one or two d- dalkyl or -C 1 -C 4 hydroxyalkyl groups.
  • the hydroxyl group in hydroxyalkyl is unsubstituted or etherified with d-C 4 alkyl.
  • alkyl examples have been given previously.
  • Examples of aminoalkyl are aminomethyl, aminoethyl, 1 -aminoprop-2-yl or -3-yl, 1 -aminobut-2-yl or -3-yl or -4-yl, N-methyl- or N,N-di- methyl- or N-ethyl- or N,N-diethyl- or N-2-hydroxyethyl- or N,N-di-2-hydroxyethylamino- methyl or -aminoethyl or -aminopropyl or -aminobutyl.
  • Examples of hydroxyalkyl are hydroxymethyl, 1-hydroxyeth-2-yl, 1 -hydroxyprop-2- or -3-yl, 1 -hydroxybut-2-yl, -3-yl or -4-yl.
  • Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxy- butyl, and examples of carbalkoxyalkyl are these carboxyalkyl groups esterified with methyl or ethyl.
  • Examples of alkenyl are allyl, but-1 -en-3-yl or -4-yl, pent-3- or 4-en-1 -yl or -2-yl, hex-3- or -4- or -5-en-1 -yl or -2-yl.
  • alkyl- and alkoxyphenyl or benzyl are methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, methylbenzyl, dimethylbenzyl, ethylbenzyl, diethylbenzyl, methoxyphenyl, dimethoxyphenyl, ethoxyphenyl, diethoxyphenyl, methoxybenzyl, dimethoxybenzyl , ethoxybenzyl, diethoxybenzyl.
  • imidazolylalkyi in which the alkyl group preferably contains 2 to 4 C atoms, are 1 ,2-, 1 ,3- or 1 ,4-imidazolylethyl or -n-propyl or -n-butyl.
  • R 15 is preferably H, methyl or ethyl.
  • Preferred examples of primary amino and secondary amino are methyl-, ethyl-, dimethyl-, diethyl-, allyl-, mono- or di(1 -hydroxyeth-2-yl)-, phenyl- and benzylamino, acetylamino, isobutyrylamino, benzoyiamino, phenoxyacetylamino, 4-tert.-butylphenoxyacetylamino,
  • R 8 and R 9 independently of one another are H, F, Cl, Br, OH, SH, NH 2 , NHOH, NHNH 2 , methyl, methylamino, dimethylamino, benzoyiamino, isobutyrylamino, methoxy, ethoxy, methylthio, phenoxyacetylamino, 4-tert.-butylphenoxyacetylamino,
  • analogues of the purine series are adenine, N-methyl- adenine, N-benzoyladenine, 2-methylthioadenine, 2-amino-6-chloropurine, 2-amino-6- methylthiopurine, 2-aminopurine, hypoxanthine, 2-aminoadenine, 6-hydroxypurine, guanine and N-isobutyrylguanine.
  • Adenine, 2-aminoadenine, 2-aminopurine, guanine and hypoxanthine are particularly preferred.
  • a or B in formula 2 is an analogous pyrimidine radical, it is preferably uracil, thymine or cytosine radicals of formulae 9 or 10
  • R 16 and R 17 independently of one another are is H, F, Cl, Br, CONH 2 , alkyl, propinyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atom; phenyl; benzyl; primary amino having 1 to 20 C atoms or secondary amino having 2 to 30 C atoms; the hydrogen atoms of the NH 2 group in formula 10 are unsubstituted or substituted by C C 6 alkyl, benzoyl or benzyl; and the dihydro derivatives of the radicals of formulae 9 and 10:
  • R 16 is preferably H, F, Cl, Br, d-dalkyl, C,-C 6 alkenyl, d-C 6 alkinyl, d-C 6 hydroxyalkyl, d- C 6 aminoalkyl, NHd-C 4 alkyl, N(d-C 4 alkyl) 2 , propinyl; and more preferably H, F, Cl, Br, methyl, ethyl, or propinyl; and most preferably H, propinyl or methyl;
  • R 17 is preferably H, F, Cl, Br, C C 6 alkyl or d-C 6 alkoxy, d-C 6 hydroxyalkyl, d-C 6 aminoalkyl, NH 2 , NHd-dalkyl, N(d-C 4 alkyl) 2 , and propinyl; and more preferably H , F, Cl, Br, methyl, ethyl, and propinyl; and most preferably H, propinyl or methyl.
  • pyrimidine analogues are uracil, thymine, cytosine, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, 5-methylcytosine, 5-propinyluracil, 5-propinylcytosine and their base protected derivatives.
  • Especially preferred structural units are of formula B8, B28 and B49.
  • nucleoside dimer of the formula 12 that can be used, for example, as a building block for the construction of oligonucieotides as shown in formula 1.
  • R 1 , R 2 , R 3 , X,Y, m, A and B are as defined above;
  • R 1 ⁇ and R 19 independent of one another are H, an OH-protecting group or a phosphorus- containing, nucleotide-bridge-group-forming radical.
  • R 18 is H or an OH-protecting group and R 19 is a phosphorus- containing, nucleotide-bridge-group-forming radical.
  • Suitable Protective groups and processes for derivatisation of the hydroxyl groups with such protective groups are generally known in sugar and nucleotide chemistry and described, for example, by B. T. Greene, Protective Groups in Organic Synthesis, Wiley Interscience, New York (1991 ).
  • Examples of such protective groups are: linear or branched d-C 8 alkyl, particularly d-dalkyl, for example methyl, ethyl, n- and i-propyl, n-, i- and t-butyl; C 7 - C ⁇ 8 aralkyl, for example benzyl, methylbenzyl, dimethylbenzyl, methoxy benzyl, dimethoxy- benzyl, bromobenzyl, diphenylmethyl, di(methylphenyl)methyl, di(dimethylphenyl)methyl, di(methoxyphenyl)methyl, di(dimethoxyphenyl)methyl, trityl, tri(methylphenyl)methyl, tri(di- methylphenyl)methyl, methoxyphenyl(diphenyl)methyl, di(methoxyphenyl)phenylmethyl, tri(dimethoxyphenyl)methyl, tri(methoxyphenyl)methyl; triphen
  • the protecting group is alkyl, it can be substituted by F, Cl, Br, C ⁇ -C 4 alkoxy, phenoxy, chlorophenoxy, methoxyphenoxy, benzyloxy, methoxybenzyloxy or chlorophenoxy.
  • the protective groups are, independently of one another, linear or branched d-C 4 alkyl, C 7 -d ⁇ aralkyl, trialkylsilyl having 1 to 12 C atoms in the alkyl groups; -(d-C 4 alkyl) 2 Si-O-Si(C C 4 alkyl) 2 like (CH 3 ) 2 Si-O-Si(CH 3 ) 2 - and -(i-C 3 H 7 ) 2 Si-O-Si(iC 3 H 7 ) 2 -; C 2 - C 8 acyl, R 12 -SO 2 -, in which R 12 is d-C 6 alkyl; phenyl or benzyl unsubstituted or substituted with F, Cl or Br; C ⁇ -C alkylphenyl; d-C alkylbenzyl; C ⁇ -C 8 alkoxycarbonyl; phenoxycarbonyl; benzyloxycarbonyl or 9-fluoreny
  • the protective groups are methyl, ethyl, n- or i- propyl, n-, i- or t-butyl; benzyl, methylbenzyl, dimethylbenzyl, methoxybenzyl, dimethoxy- benzyl, bromobenzyl; diphenylmethyl, di(methylphenyl)methyl, di(dimethylphenyl)methyl, di(methoxyphenyl)methyl, di(methoxyphenyl)(phenyl)methyl, trityl, tri(methylphenyl)methyl, tri(dimethylphenyl)methyl, tri(methoxyphenyl)methyl, tri(dimethoxyphenyl)methyl; trimethyl- silyl, triethylsilyl, tri-n-propylsilyl, i-propyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl
  • R 18 is
  • R 19 is CH 3 CH 3
  • a phosphorus-containing, nucleotide-bridge-group-forming radical may correspond to formula P1 or P2
  • Y a is hydrogen, C ⁇ -C ⁇ 2 alkyl, C 6 -C 12 aryl, C 7 -C 20 aralkyl, C 7 -C 2 oalkaryl, -OR b , -SR b , secondary amino, O * M + or S * M + ;
  • X a is oxygen or sulfur;
  • R a is hydrogen, M + , d-C 12 alkyl, C 2 -C 12 alkenyl or C 6 -C ⁇ 2 aryl, or the group R a O- is N-hetero- aryl-N-yl having 5 ring members and from 1 to 3 nitrogen atoms;
  • R b is hydrogen, C ⁇ -C 2 alkyl or C 6 -d 2 aryl;
  • M + is Na + , K + , Li + , NH 4 + or primary, secondary, tertiary or quaternary ammonium; alkyi, aryl, aralkyl and alkaryl in Y a , R a and R b being unsubstituted or substituted by alkoxy, alkylthio, halogen, -CN, -NO 2 , phenyl, nitrophenyl or halophenyl.
  • Y a contains as secondary amino preferably from 2 to 12 and especially from 2 to 6 carbon atoms.
  • the secondary amino may be, for example, a radical of the formula R c RdN, wherein R c and R d , are independently of one another is C ⁇ -C 20 -, preferably d-C 12 - and especially C C 6 - alkyl; d-C 20 -, preferably C ⁇ -C 12 - and especially Ci-d-aminoalkyl; or C ⁇ -C 2 o-, preferably d- C 12 - and especially d-C 6 -hydroxyalkyl; carboxyalkyl or carbalkoxyalkyi, the carbalkoxy group containing from 2 to 8 carbon atoms and the alkyl group from 1 to 6, preferably from 1 to 4, carbon atoms; C 2 -C 20 -, preferably C 2 -C 12 - and especially C 2 -C 6 -alkenyl; phenyl, mono- or di-(C ⁇ -C 4 alkyl or d-C 4 alkoxy)phenyl, benzyl, mono
  • C ⁇ -C 20 - preferably d-C ⁇ 2 - and especially C ⁇ -C 6 -alkyl, d-C 20 -, preferably d-C 12 - and especially d-C ⁇ -aminoalkyl, C ⁇ -C 2 o-, preferably d-C 12 - and especially C C 6 -hydroxyalkyl; carboxyalkyl or carbalkoxyalkyi, the carbalkoxy group containing from 2 to 8 carbon atoms and the alkyl group from 1 to 6, preferably from 1 to 4, carbon atoms; C 2 -C 20 -, preferably C 2 - d 2 - and especially C 2 -C 6 -alkenyl; phenyl, mono- or di-(d-C 4 alkyl or d-C 4 alkoxy)phenyl, benzyl, mono- or di-(d-C 4 alkyl or C ⁇ -C 4 alkoxy)benzyl; or 1 ,2-, 1 ,3- or
  • Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxy- butyl, and examples of carbalkoxyalkyi are those carboxyalkyl groups esterified by methyl or ethyl.
  • Examples of alkenyl are allyl, but-1-en-3-yl or -4-yl, pent-3- or -4-en-1 -yl or -2-yl, hex- 3- or -4- or -5-en-1 -yl or -2-yl.
  • alkyl- and alkoxy-phenyl and alkyl- and alkoxy- benzyl are methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, methylbenzyl, dimethylbenzyl, ethylbenzyl, diethylbenzyl, methoxyphenyl, dimethoxyphenyl, ethoxyphenyl, diethoxyphenyl, methoxybenzyl, dimethoxybenzyl, ethoxybenzyl and diethoxybenzyl.
  • Examples of imidazolylalkyi in which the alkyl group preferably contains from 2 to 4 carbon atoms are 1 ,2-, 1 ,3- or 1 ,4- ⁇ midazolyl-ethyl or -n-propyl or -n-butyl.
  • R e is preferably hydrogen, methyl or ethyl.
  • Preferred examples of primary ammo and secondary amino are methyl-, ethyl-, dimethyl-, diethyl-, diisopropyl, mono- or d ⁇ -(1 -hydroxy-eth-2-yl)-, phenyl- and benzyl-ammo, acetyl- amino and benzoyiamino and pipendinyl, piperazinyl and morpholinyl
  • Preferred examples of primary and secondary ammonium are methyl-, ethyl-, dimethyl-, diethyl-, diisopropyl-, mono- or d ⁇ -(1 -hydroxy-eth-2-yl)-, phenyl- and benzyl-ammonium
  • Preferred substituents are chlorine, bromine, methoxy, -NO 2 , -CN, 2,4-dichlorophenyl and 4-n ⁇ trophenyl.
  • R b are 2,2,2-tr ⁇ chloroethyl, 4-chlorophenyl, 2-chlorophenyl and 2,4-d ⁇ chlorophenyl; and examples of R b O- as N-heteroaryl are pyrrol-N-yl, t ⁇ azol-N-yl and benzot ⁇ azol-N-yl.
  • R a is ⁇ -cyanoethyl and Y a is d ⁇ ( ⁇ sopropylam ⁇ no) ln an especially preferred form the dinucleoside analog is of formula C8, C28 or C49
  • the invention further relates to a process for the preparation of compounds of the formula 12, which is characterized in that a compound of the formula 14
  • R 1 , X and A are as defined above;
  • R 27 is H or an OH-protecting group as defined above.
  • R 29 is H or an ester activating group like C 6 F 5 , p-NO 2 -phenyl, hydroxybenzotriazol-1 -yl and
  • R 2 , R 3 , Y and B are as defined above;
  • a condensing agent like, e.g., dicyclohexylcarbodi- imide, TBTU (benzotriazol-1 -yl-tetramethyluronium tetrafluoroborate) or HBTU (hexa- fluorophosphate).
  • the temperature in the synthesis reaction can be from -80 to 150°C, preferably 0 to 100°C.
  • solvents which are protic and/or aprotic, and particularly preferably dipolar.
  • solvents which can be employed on their own or as a mixture of at least two solvents are ethers (dibutyl ether, tetrahydrofuran, dioxane, diethylene glycol dimethyl ether, ethylene glycol dimethyl or diethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether), halogenated hydrocarbons (methylene chloride, chloroform, 1 ,2- dichloroethane, 1 ,1 ,1 -trichloroethane, 1 ,1 ,2,2-tetrachloroethane), carboxylic acid esters and lactones (ethyl acetate, methyl propionate, ethyl benzoate, 2-methoxyethy!
  • An object of the present invention is the use of a dimer of formula 12 for the preparation of oligonucieotides which comprise one or more identical or different dimer units of formula 12.
  • oligonucieotides according to the invention can be prepared in a manner known per se by various processes, preferably on a solid support. For details see for example Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984).
  • the oligonucieotides of the formula 1 and the dimeres of formula 12 can be used in a method of treatment. They have, e.g., antiviral and antiproliferative properties.
  • the oligonucieotides and dimeres according to the invention have a surprisingly high stability to degradation by nucieases. A very good pairing with complementary nucleic acid strands, particularly of the RNA type, is also observed.
  • the oligonucieotides according to the invention are therefore particularly suitable for antisense technology, i.e.
  • oligonucieotides according to the invention are also suitable as diagnostics and can be used as gene probes for the detection of viral infections or of genetically related diseases by selective interaction at the single- or double-stranded nucleic acid stage.
  • the invention relates to the use of the oligonucieotides according to the invention as diagnostics for the detection of viral infections or of genetically related diseases.
  • the invention also relates to the oligonucieotides of the formula 1 and dinucleosides of formula 12, according to the invention, for use in a therapeutic process for the treatment of diseases in mammals including humans by means of inactivation of nucleotide sequences in the body.
  • the dose when administered to mammals of about 70kg body weight can be, for example, 0.01 to 1000mg per day.
  • Administration is preferably effected parenterally, for example intravenously or intraperitoneally, in the form of pharmaceutical preparations.
  • the invention further relates to a pharmaceutical preparation comprising an effective amount of an oligonucleotide of the formula 1 or dimeres of formula (12) on its own or together with other active ingredients, a pharmaceutical carrier in a customary amount and, if appropriate, excipients.
  • the pharmacologically active oligonucieotides or dimeres according to the invention can be used in the form of parenterally administrable preparations or of infusion solutions. Solutions of this type are preferably isotonic aqueous solutions or suspensions, it being possible to prepare these before use, for example in the case of lyophilized preparations which contain the active substance on its own or together with a carrier, for example mannitol.
  • the pharmaceutical preparations can be sterilized and/or contain excipients, for example preservatives, stabilisers, wetting and/or emulsifying agents, solubilisers, salts for regulating the osmotic pressure and/or buffers.
  • the pharmaceutical preparations which if desired can contain further pharmacologically active substances such as, for example, antibiotics, are prepared in a manner known per se, for example by means of conventional dissolving or iyophilizing processes, and contain about 0.1 % to 90%, in particular from about 0.5% to about 30%, for example 1 % to 5% of active substance(s).
  • TTTr tris tert. butyl trityl
  • A4: 1 H NMR (500 MHz, CDCI 3 ): ⁇ 3.70 dd, 1 H, H-C(4')); MS(EI): 494 (M+H)
  • Alcohol A7 (108 mg, 0.130 mmol), dissolved in CH 2 CI 2 (2ml), is added to a solution of di- isopropylammon i um tetrazolide ( 1 5 mg, 0.088 mmol) and cyanoethoxy-bis- diisopropylamino-phosphine (58 mg, 0.195 mmol) in CH 2 CI 2 (2 ml) at 25°C.
  • reaction mixture is stirred for 3 h, poured into aqueous, saturated NaHCO 3 -solution, extracted with CH 2 CI 2 (3x), the organic layers are washed with brine, dried (Na 2 SO 4 ), concentrated, and purified by flash chromatography (1 -10 % MeOH in EtOAc, 1 % Et 3 N) to give compound A8 (120 mg, 90 %).
  • Alcohol A27 300 mg, 0.339 mmol
  • di-isopropylammonium tetrazolide 437 mg, 2.55 mmol
  • HV 12 h
  • CH 2 CI 2 10 ml
  • cyanoethoxy-bis- diisopropylamino-phosphine 460 mg, 1.53 mmol
  • the reaction mixture is stirred for 6 h at 25°C.
  • the reaction is concentrated, dissolved in CH 2 CI 2 and precipitated in cold pentane.
  • the mother liquor is concentrated and remaining product is precipitated.
  • a solution of compound A34 (6.0 g, 17.2 mmol) in 90% AcOH (90 mi) is stirred for 5 h at 80°C and 16 h at 25°C, cooled to 0°C and carefully treated with solid NaHCO 3 .
  • the reaction mixture is concentrated, coevaporated with toluene (2x) and purified by flash chromatography (silica, 35-50% EtOAc in hexane) to give A35 (5.2 g, 98%).
  • reaction mixture is poured into saturated, aqueous NaHCO 3 solution, extracted with CH 2 CI 2 (3x), the combined organic layers are washed with brine, dried (Na 2 SO 4 ), concentrated and purified by flash chromatography (50% EtOAc in hexane) to give compound A43 (1.91 , 94%).
  • Alcohol A49 (338 mg, 0.355 mmol), dissolved in CH 2 CI 2 (5ml), is added to a solution of di- isopropylammonium tetrazohde (67 mg, 0.0.391 mmol) and cyanoethoxy-bis-dnsopropyl- amino-phosphine (235 mg, 0.78 mmol) in CH 2 CI 2 (10 ml) at 25°C.
  • reaction mixture is stirred at RT for 2 h and is then poured into aqueous, saturated NaHCO 3 -solution, extracted with CH 2 CI 2 (3x), the organic layers are washed with brine, dried (Na 2 SO ), concentrated, and purified by flash chromatography (2-10 % MeOH in EtOAc, 1% Et 3 N) to give compound A50 (366 mg, 88 %).
  • Alcohol A53 (315 mg, 0.38 mmol), dissolved in CH 2 CI 2 (2ml), is added to a solution of di- isopropylammonium tetrazolide (44 mg, 0.256 mmol) and cyanoethoxy-bis- diisopropylamino-phosphine (172 mg, 0.0.573 mmol) in CH 2 CI 2 (2 ml) at 25°C.
  • reaction mixture is stirred for 5 h, poured into aqueous, saturated NaHCO 3 -solution, extracted with CH 2 CI 2 (3x), the organic layers are washed with brine, dried (Na 2 SO 4 ), concentrated, and purified by flash chromatography (1-10 % MeOH in EtOAc, 1 % Et 3 N) to give compound A54 (365 mg, 93 %).
  • Each oligonucleotide is prepared on an ABI 390 DNA synthesizer using standard phos- phoramidite chemistry according to Gait, M.J., Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984) but with prolonged coupling times (10 min).
  • Dimeth- oxytrityl oligonucieotides are purified by reverse phase HPLC (column: Nucleosil RPC 18 , 10 ⁇ , 10x 250 mm; eluent A: 50 mM triethylammonium acetate (TEAA), pH 7.0; eluent B: 50mM TEAA, pH 7.0 in 70 % acetonitrile; elution with gradient from 15 % to 45 % B in 45 min).
  • TEAA triethylammonium acetate
  • eluent B 50mM TEAA, pH 7.0 in 70 % acetonitrile
  • the oligodeoxynucleotides are controlled by capillary gel electrophoresis (concentration: 1 OD/ml, injection: 2 kV, 3 sec, separation: 9kV, capillary: effective length 30 cm, inner diameter 100 ⁇ m, polyacryiamide 10 % T, buffer: 100 mM H 3 PO 4 , 100 mM Tris, 2 mM EDTA, 7 M urea pH 8.8) .
  • the molecular weight of each oligodeoxynucleotide is checked by mass spectroscopy [MALDI-TOF: Pieles, U., Z ⁇ rcher, W., Schar, M., Moser, H., Nucl. Acids Res.
  • the oligodeoxynucleotide is desorbed using 2,4,6-trihydroxyacetophenone as a matrix (detection of negatively charged ions) with diammonium hydrogen citrate as additive (25mM final concentration).
  • T m The thermal denaturation (T m ) of DNA/RNA hybndes is performed at 260 nm using a Gilford Response II Spectrophotometer (Ciba-Corning Diagnostics Corp. , Oberlm, OH) . Absorbance versus temperature profiles are measured at 4 ⁇ M of each strand in 10 mM phosphate pH 7.0 (Na salts), 100 mM total [Na + ] (supplemented as NaCl), 0.1 mM EDTA.
  • T m 's are obtained from fits of absorbance versus temperature curves to a two-state model with linear slope baselines [Freier, S.M., Albergo, D.D., Turner, D.H., Biopolymers 22:1 107- 1 131 (1982)]. All values are averages of at least three experiments. The absolute experimental error of the T m values is ⁇ 0.5°C.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention porte notamment sur un oligonucléotide défini par la formule (1) 5'-(U)n-3', dans laquelle U représente des radicaux, identiques ou différents, d'un nucléoside naturel ou synthétique. Cet oligonucléotide comporte au moins un nucléotide dimère modifié composé de deux analogues de nucléosides reliés par une liaison amide présentant une configuration particulière. L'invention concerne également la synthèse de ces composés et leur utilisation dans des préparations pharmaceutiques.
PCT/EP1997/003192 1996-06-28 1997-06-19 Oligonucleotides modifies WO1998000434A1 (fr)

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EP96810431.5 1996-06-28

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WO2010048549A2 (fr) * 2008-10-24 2010-04-29 Isis Pharmaceuticals, Inc. Nucléotides bis-substitués en 5’ et 2’ et composés oligomères préparés à partir de ceux-ci
US8623901B2 (en) 2009-03-31 2014-01-07 Boehringer Ingelheim International Gmbh Compounds for the treatment of CNS disorders
US8623879B2 (en) 2008-04-02 2014-01-07 Boehringer Ingelheim International Gmbh 1-heterocyclyl-1,5-dihydro-pyrazolo[3,4-D] pyrimidin-4-one derivates and their use as PDE9A modulators
US8648085B2 (en) 2007-11-30 2014-02-11 Boehringer Ingelheim International Gmbh 1, 5-dihydro-pyrazolo (3, 4-D) pyrimidin-4-one derivatives and their use as PDE9A mudulators for the treatment of CNS disorders
US8809345B2 (en) 2011-02-15 2014-08-19 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8912201B2 (en) 2010-08-12 2014-12-16 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US8987435B2 (en) 2008-10-24 2015-03-24 Isis Pharmaceuticals, Inc. Oligomeric compounds and methods
US8993738B2 (en) 2010-04-28 2015-03-31 Isis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
US9067945B2 (en) 2002-08-23 2015-06-30 Boehringer Ingehleim International GmbH Selective phosphodiesterase 9A inhibitors as medicaments for improving cognitive processes
US9079905B2 (en) 2008-09-08 2015-07-14 Boehringer Ingelheim International Gmbh Compounds for the treatment of CNS disorders
WO2018130848A1 (fr) * 2017-01-13 2018-07-19 Oxford University Innovation Limited Ligature d'oligonucléotides
US10676738B2 (en) 2010-04-28 2020-06-09 Ionis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom

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US9067945B2 (en) 2002-08-23 2015-06-30 Boehringer Ingehleim International GmbH Selective phosphodiesterase 9A inhibitors as medicaments for improving cognitive processes
US8648085B2 (en) 2007-11-30 2014-02-11 Boehringer Ingelheim International Gmbh 1, 5-dihydro-pyrazolo (3, 4-D) pyrimidin-4-one derivatives and their use as PDE9A mudulators for the treatment of CNS disorders
US9096603B2 (en) 2008-04-02 2015-08-04 Boehringer Ingelheim International Gmbh 1-heterocyclyl-1,5-dihydro-pyrazolo[3,4-D] pyrimidin-4-one derivatives and their use as PDE9A modulators
US8623879B2 (en) 2008-04-02 2014-01-07 Boehringer Ingelheim International Gmbh 1-heterocyclyl-1,5-dihydro-pyrazolo[3,4-D] pyrimidin-4-one derivates and their use as PDE9A modulators
US9079905B2 (en) 2008-09-08 2015-07-14 Boehringer Ingelheim International Gmbh Compounds for the treatment of CNS disorders
US8883752B2 (en) 2008-10-24 2014-11-11 Isis Pharmaceuticals, Inc. 5′ and 2′ BIS-substituted nucleosides and oligomeric compounds prepared therefrom
WO2010048549A3 (fr) * 2008-10-24 2010-09-10 Isis Pharmaceuticals, Inc. Nucléotides bis-substitués en 5’ et 2’ et composés oligomères préparés à partir de ceux-ci
WO2010048549A2 (fr) * 2008-10-24 2010-04-29 Isis Pharmaceuticals, Inc. Nucléotides bis-substitués en 5’ et 2’ et composés oligomères préparés à partir de ceux-ci
US9738895B2 (en) 2008-10-24 2017-08-22 Ionis Pharmaceuticals, Inc. Oligomeric compounds and methods
US8987435B2 (en) 2008-10-24 2015-03-24 Isis Pharmaceuticals, Inc. Oligomeric compounds and methods
US8623901B2 (en) 2009-03-31 2014-01-07 Boehringer Ingelheim International Gmbh Compounds for the treatment of CNS disorders
US9102679B2 (en) 2009-03-31 2015-08-11 Boehringer Ingelheim International Gmbh Compounds for the treatment of CNS disorders
US11268094B2 (en) 2010-04-28 2022-03-08 Ionis Pharmaceuticals, Inc 5′ modified nucleosides and oligomeric compounds prepared therefrom
US8993738B2 (en) 2010-04-28 2015-03-31 Isis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US11084844B2 (en) 2010-04-28 2021-08-10 Ionis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US10676738B2 (en) 2010-04-28 2020-06-09 Ionis Pharmaceuticals, Inc. 5′ modified nucleosides and oligomeric compounds prepared therefrom
US9321799B2 (en) 2010-04-28 2016-04-26 Ionis Pharmaceuticals, Inc. Modified nucleosides, analogs thereof and oligomeric compounds prepared therefrom
US8912201B2 (en) 2010-08-12 2014-12-16 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
US9328120B2 (en) 2010-08-12 2016-05-03 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US9278990B2 (en) 2010-09-22 2016-03-08 Alios Biopharma, Inc. Substituted nucleotide analogs
US8809345B2 (en) 2011-02-15 2014-08-19 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
US9605018B2 (en) 2011-12-22 2017-03-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US9394330B2 (en) 2012-03-21 2016-07-19 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US9856284B2 (en) 2012-03-21 2018-01-02 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
WO2018130848A1 (fr) * 2017-01-13 2018-07-19 Oxford University Innovation Limited Ligature d'oligonucléotides

Also Published As

Publication number Publication date
AU3340797A (en) 1998-01-21
ZA975742B (en) 1997-12-29

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